SOLICITACIONES PARA PUENTES

Protein folding intermediates are usually more compact than the unfolded state but less compact than the native state. They often have hydrophobic surface patches which can result in an increased ten- dency to aggregate. Detailed understanding of the effects that lead to the aggregation of these partially

2_{This is more than expected for a 4 M GuHCl solution (1.27x, Kawahara and Tanford, 1966). This deviation may be}

caused by a change in focus size due to the higher refractive index of the GuHCl solution. Using the dye as a reference allows taking both viscosity change and refractive index change into account (Enderleinet al., 2005).

(<1 µs) during folding which can make observation difficult. However, stable folding intermediates have often been observed at acidic conditions and moderate ionic strength (A-states, Fink, 1995). One model system for the investigation of the A-state is the acidic compact state of apomyoglobin from sperm whale. An increasing number of spectroscopic techniques, particularly NMR (Barrick and Baldwin, 1993; Cocco and Lecomte, 1990), has been employed to understand the nature of the acidic compact state. However, the partially unfolded ensemble of proteins is actually comprised of a large number of structurally different states, and ensemble spectroscopic techniques like NMR will provide measurements only for the average properties of the system.

MD simulations have been employed to characterize the states comprising the acidic compact state of yellowfin tuna myoglobin in molecular detail (Bismutoet al., 2009). To validate the results of the MD simulations, several spectroscopic methods were employed. The presence of a single Tryptophan in yellowfin tuna apomyoglobin allows an easier interpretation of its intrinsic fluorescence. The lifetime distribution of the Tryptophan residue measured by Frequency Domain Fluorometry broadens for lower pH suggesting more heterogeneity of myoglobin in the A-state compared to the native state. The acrylamid quenching data is in good agreement with the collisional quenching constant of the intrinsic fluorescence emission of the 25 MD-generated acidic conformers of myoglobin. Also, the α-helical content measured by circular dichroism is in very good agreement with the MD simulations. The diffusion coefficients obtained by 2fFCS measurements are compatible with the calculated ones taking the protein hydration shell into account. While the agreement at pH 7 is very good, the measured values at pH 3 are somehow larger than expected from the MD simulations. However, taking the uncertainties of both the MD simulations (approximation of the force field parameters, cut off, periodic conditions, and a limited number of generated structures) and the 2fFCS measurements (reference diffusion coefficient of Rhodamine 6G, possible aggregation at acidic pH) into account, these differences are within the experimental errors.

Chenet al.(2007) have studied the fast fluctuations of myoglobin in various pH caused by quench- ing of an N-terminal dye by aromatic residues using FCS. As a byproduct of these measurements, they also report on changes of the diffusion coefficient with varying pH. They also observed a pH- dependent 40% change ofD, however, in their measurements,Dis essentially pH independent be- tween pH 4.5 and 7 and the transition midpoint is lower than pH 3.5. The difference to the pH dependence in the 2fFCS experiments presented here may be explained by the use of sperm whale myoglobin by Chenet al.(2007) or the ionic strength differences between the two studies.

In conclusion, the good agreement of the experimental results, in particular the 2fFCS results, show the validity of the structures derived from the MD simulations. Combined, the simulation and exper- imental results support the point of view that folding intermediates cannot be described by a single conformational state but rather by a number of structurally different conformations with similar en- ergetic characteristics. A subset of these states shows exposed hydrophobic surface patches that possibly can trigger the aggregation of the protein.

Some results of this chapter have been published previously in (Mapaet al., 2010).

6.1 Introduction

6.1.1 The Hsp70 chaperones

Heat shock proteins 70 (Hsp70s) are a highly conserved and ubiquitous class of molecular chaper- ones present in prokaryotic organisms and in all major organelles of eukaryotes. They are involved in folding of newly synthesized proteins, prevention of protein aggregation, remodeling of protein com- plexes and transport of proteins across membranes. These functions are performed by ATP-regulated binding and release of substrate proteins.

All Hsp70s share a similar structure (Figure 6.1). They consist of an N-terminal 44 kDa nucleotide binding domain (NBD) connected by a flexible linker to the C-terminal 25 kDa substrate binding domain (SBD). The SBD is divided into a β-sandwich sub-domain with a hydrophobic cleft for substrate binding and an α-helical lid. The SBD binds hydrophobic regions of substrate peptides. The affinity of the SBD for substrate is regulated by the binding of different nucleotides to the NBD. In the ATP-bound state, the lid is open and the affinity for substrate is low. ATP hydrolysis and substrate binding is stimulated by J proteins. Then, release of ADP and subsequent ATP binding is modulated by nucleotide exchange factors (NEF), allowing substrate release and the beginning of a new cycle (Bukauet al., 2006; Frydman, 2001; Genevauxet al., 2007; Mayer and Bukau, 2005; Saibil, 2008; Younget al., 2004).

The structures of the substrate-bound state and the ATP-bound state of different Hsp70s have been recently solved using NMR and X-ray crystallography (for an overview, refer to Bertelsenet al., 2009, and references therein). However, these structures are only static pictures of specific states of the Hsp70 cycle and do not give insight into the processes involved in the functional cycle. The dynamic processes connecting these states can be resolved by single-molecule fluorescence studies using spFRET. However, only few studies have used single-molecule fluorescence to get more insight on the function of Hsp70s. Besides the work on mtHsp70 presented in this thesis and in (Mapa

et al., 2010), another paper (Marcinowskiet al., 2011) studied the only Hsp70 in the endoplasmatic reticulum, the Immunoglobulin Binding Protein (BiP). Marcinowski et al. (2011) used a spFRET assay to study the conformation of BiP in different stages of its conformational cycle. The labeling positions were designed in a similar way as in this work and one additional sensor monitored the distance between the tip of the substrate binding lid and the NBD. In the apo (nucleotide-free) state and in the ADP state, BiP has a high conformational flexibility. ATP binding results in domain

Figure 6.1:The structure of Hsp70 chaperones. The two domains are denoted in a sketch of a general Hsp70 (left) and the structure of DnaK (PDB: 2KHO).

docking and opening of the substrate binding lid. Substrate binding does not result in further domain undocking and lid closing compared to the ADP state. The conformation of BiP changes significantly depending on the substrate bound. It is similar to the ATP state for BiP bound to the complete first domain of the IgG heavy chain CH1 and similar to the ADP state for a small peptide derived from

CH1 suggesting substrate discrimination by the lid communicated to the NBD.

6.1.2 Mitochondrial Hsp70

In the mitochondrial matrix of yeast, three different species of Hsp70s with significant sequence ho- mology to the bacterial Hsp70 DnaK are present. Ssq1 is involved in the biogenesis of Fe-S clusters, and Ecm10 is of low abundance and has been suggested to have a similar function as Ssc1 (Bau- mannet al., 2000; Craig and Marszalek, 2002; Voos and Röttgers, 2002). The major mitochondrial Hsp70, known as Ssc1, mediates folding of proteins in the mitochondrial matrix and is involved in the import of preproteins into the mitochondrial matrix. Both the protein folding and the preprotein translocation activities of Ssc1 rely on its affinity for unfolded proteins and its ATPase activity. Ssc1 makes up 1-3 % of mitochondrial protein and is essential for cell viability (Craiget al., 1987; Craig and Marszalek, 2002; Yonedaet al., 2004). A specialized chaperone, Hep1, is required to maintain the structure and function of Ssc1 (Sichtinget al., 2005).

6.1.2.1 Role of Ssc1 in protein import into mitochondria

Only few of the proteins present in mitochondria are actually produced there. The vast majority is produced by cellular ribosomes and has to be imported into mitochondria and targeted to the correct sub-compartment. Several translocation machineries exist in mitochondria to correctly deliver the preprotein to its destination (Neupert and Brunner, 2002; Mokranjac and Neupert, 2009). Prepro- teins are imported through the outer mitochondrial membrane to the inter-membrane space by the TOM complex. Then, the translocase of the inner mitochondrial membrane 23 (TIMM23) complex

membrane potential across the inner mitochondrial membrane and from ATP hydrolysis.

The translocation channel is formed by the channel-forming membrane proteins Tim23 and Tim17. Targeting of the preprotein to the outer part of the channel is mediated by the receptor protein Tim50. The protein Tim44 resides associated to the membrane close to the translocation channel and is responsible for the recruitment of the soluble components of the import motor. The actual import motor of the translocation complex is the mitochondrial Hsp70 Ssc1 present in the mitochondrial matrix in conjunction with the J protein Tim14, the J-like protein Tim16 (Bolender et al., 2008; Craiget al., 2006; Mokranjacet al., 2006), and the nucleotide exchange factor Mge1 (Neupert and Brunner, 2002).

The positively charged targeting sequence of precursor proteins is transported through the translo- cation pore by the potential across the inner mitochondrial membrane (Neupert and Brunner, 2002). Then, Ssc1 can bind to hydrophobic parts of the preprotein chain and an inward motion of the protein is generated. Generally, motor proteins can promote this motion according to two different princi- ples. First, brownian motion may be the driving force and the motor protein prevents backsliding passively by binding to the preprotein (brownian ratchet model). Second, the force responsible for the inward motion can be generated by an active conformational change of the motor protein, with ATP hydrolysis as the source of the energy (power stroke model). For Ssc1, a third model has been proposed combining both ideas into anentropic pulling model. This model suggests that the limited amount of freedom of motion for Ssc1 close to the membrane induces an entropic force in the inward direction (De Los Rioset al., 2006; Goloubinoff and De Los Rios, 2007).

6.1.2.2 Role of Ssc1 in protein folding in the mitochondrial matrix

Like the majority of Hsp70 chaperones, Ssc1 supports folding of proteins and prevents their aggre- gation, in addition to its function as an import motor (Horstet al., 1997; Kanget al., 1990; Rowley

et al., 1994; Westermannet al., 1996). In this function, it is supported by the J protein Mdj1 nd the nucleotide exchange factor Mge1. The chaperone activity of Ssc1 has been demonstrated by in vitro experiments, where a reconstituted system including Ssc1, Mdj1 and Mge1 was able to prevent the aggregation and increase the refolding efficiency of heat-denatured luciferase (Westermann et al., 1996; Kuboet al., 1999).

Although the protein folding cycle of Ssc1 is closely related to the cycle of the bacterial Hsp70 DnaK and Ssc1 and DnaK share over 50% sequence identity, yeast Ssc1 and DnaK from E. coli

cannot substitute each other, neither in bacteria nor in yeast mitochondria (Deloche et al., 1997; Moroet al., 2002). In contrast, the respective nucleotide exchange factors, Mge1 for Ssc1 and GrpE for DnaK, and J proteins, Mdj1 for Ssc1 and DnaJ for DnaK, can substitute for each other in both organisms, at least to a certain extent (Delocheet al., 1997; Lisse and Schwarz, 2000). Similarily, specifity of Hsp70s from different subcellular compartments of the same organism was shown which is surprising due to the high level of sequence conservation (Brodsky, 1996; Genevauxet al., 2007; Hennessyet al., 2005; Walshet al., 2004).

6.1.3 Scope of this project

Due to its wide range of different functions, Ssc1 is an essential protein for cell viability. Although many studies have elucidated the function of Ssc1 and the relation to other Hsp70s, a detailed mech-

anistic understanding of the mechanisms of Ssc1-mediated protein folding and protein import is still lacking. The inherent instability of Ssc1 prohibits the study of its conformation using crystalliza- tion methods. To this end, FRET studies can give new insights into the different conformations of Ssc1 along its functional cycle. In particular spFRET experiments may give additional insights into possible substates and inhomogeneities as well as dynamic changes.

To this end, we developed two FRET-based sensors and used them to follow the effects of nucleotides and interacting proteins on the relative distance between the two domains of Ssc1 and the opening of the SBD. The spFRET data collected by MFD-PIE experiments was analyzed using PDA to extract information about the number, mean and width of the distance distributions underlying the spFRET histograms. The dynamics of the system were followed by spFRET measurements of immobilized molecules using TIRF microscopy.

Cochaperones modulate the function of Ssc1 depending on the specific task at hand. The FRET sensors are used to study the effects of different cochaperones from the folding cycle and from the import cycle on the conformation of Ssc1. Additionally, we use similar FRET-based sensors for DnaK, the major bacterial Hsp70, to study the differences between Hsp70s with different functions.